The present methods relate generally to the field of identifying nucleic acids. In particular, the present methods relate to the field of identifying nucleic acids in a sample by detecting multiple signals such as signals emitted from fluorophores. The present methods also relate to the field of identifying nucleic acid in a sample by using labeled oligonucleotides to detect the nucleic acid in combination with agents for determining the melting temperature of the detected nucleic acid.
Methods for detecting nucleic acids such as multiplex methods are increasing important in medical diagnostics. Typical multiplex methods utilize PCR amplification, and in particular, real-time quantitative PCR. Real-time or end-point detection methods for PCR typically are based on one of two principles for monitoring amplification products: (1) specific hybridization by probes or primers to single-stranded DNA; or (2) binding by small molecules (e.g., intercalating agents) to double-stranded DNA. Probes and primers may include Molecular Beacon Probes, Scorpion® Primers, Taqman® Probes, and other labeled primers or probes. Small molecules that bind to DNA may include intercalators (e.g., SYBR™ Green I dye and ethidium bromide) and minor groove binders.
Methods for detecting nucleic acid that utilize probes and primers typically involve labeling each probe or primer with a unique label (e.g., a fluorescent dye). Because multiplexing methods commonly utilize fluorescent dyes, they often are called “color multiplexing” methods. These types of multiplexing methods are limited by the practical number of labels that can be detected and distinguished in a reaction mixture. When fluorescent dyes are used in these methods, typically no more than four (4) different dyes can be detected and distinguished based on the dyes' specific absorption (excitation) maximum wavelengths and emission maximum lengths using current detection technology.
Methods for detecting nucleic acid that utilize small binders such as intercalators typically involve determining melting temperatures (“Tm”) to detect a target nucleic acid. As such, multiplexing methods that utilize this technology often are called “Tm multiplexing” methods. These methods may include determining the melting temperature of a complex formed by a probe and the amplified target nucleic acid, or determining the melting temperature of the amplified target nucleic acid itself (i.e., the amplicon). Where an intercalator is utilized, typically the intercalator exhibits a change in fluorescence based on whether the detected nucleic acid is double-stranded or single-stranded. These methods may be limited in that intercalating agents interact with double-stranded nucleic acids non-specifically, and as such, multiple detected products must be distinguished by criteria such as resolvable melting temperatures.
Methods for detecting a nucleic acid have been described in which a fluorescent intercalator is used as a donor for fluorescence resonance energy transfer (FRET) together with a fluorescently labeled oligonucleotide probe as an acceptor for FRET. See Howell et al., “iFRET: An Improved Fluorescence System for DNA-Melting Analysis,” GENOME RESEARCH 12:1401-1407 (2002). However, these described methods require that the intercalator and the fluorescently labeled oligonucleotide probe be selected to permit FRET. Further, these methods do not involve directly observing a signal from an intercalator as an indication of the melting temperature of an amplified nucleic acid (i.e., an amplicon). Rather, iFRET involves indirectly observing a signal from an intercalator (via FRET) as an indication of the melting temperature of a complex formed between the amplified nucleic acid and a fluorescently labeled oligonucleotide probe.
By combining detection methods based on color and melting temperature (e.g., color multiplexing and Tm multiplexing methods), it may be possible to detect a plurality of nucleic acids in a sample, Such methods may be useful in diagnostic methods related to identifying human pathogens (e.g., viruses that may include respiratory pathogens such as human parainfluenza virus).